Changes in blood-brain barrier (BBB) functionality have been implicated in Parkinson's disease. This study aimed to investigate BBB transport of L-DOPA transport in conjunction with its intra-brain conversion, in both control and diseased cerebral hemispheres in the unilateral rat rotenone model of Parkinson's disease.Ex-vivo brain tissue was analyzed for changes in tyrosine hydroxylase staining as a biomarker for Parkinson's disease severity. Data were analysed by population pharmacokinetic analysis (NONMEM) to compare BBB transport of L-DOPA in conjunction with the conversion of L-DOPA into DOPAC and HVA, in control and diseased cerebral hemisphere.In Lewis rats, at 14 days after unilateral infusion of rotenone into the medial forebrain bundle, L-DOPA was administered intravenously . Serial blood samples and brain striatal microdialysates were analysed for L-DOPA, and the dopamine metabolites DOPAC and HVA. Plasma pharmacokinetics of L-DOPA could be described by a 3-compartmental model. In rotenone responders (71%), no difference in L-DOPA BBB transport was found between diseased and control cerebral hemisphere. However, in the diseased compared with the control side, basal microdialysate levels of DOPAC and HVA were substantially lower, whereas following L-DOPA administration their elimination rates were higher.Parkinson's disease-like pathology, indicated by a huge reduction of tyrosine hydroxylase as well as by substantially reduced levels and higher elimination rates of DOPAC and HVA, does not result in changes in BBB transport of L-DOPA. Taking the results of this study and that of previous ones, it can be concluded that changes in BBB functionality are not a specific characteristic of Parkinson's disease, and cannot account for the decreased benefit of L-DOPA at later stages of Parkinson's disease. Tyrosine is usually considered as the starting point in the biosynthesis of dopamine (DA). It is taken up into the brain and subsequently from brain extracellular fluid into dopaminergic neurons where its is converted to 3,4-dihydroxyphenylalanine (L-DOPA), by tyrosine hydroxylase (TH). Aromatic amino acid decarboxylase (AADC) then converts L-dopa to DA and stored in vesicles for neurotransmission . DopaminIt is known that in Parkinson's disease dopaminergic neurons in the nigro-striatal pathway are progressively damaged , which cet al. [The question whether the BBB is affected in Parkinson's disease is still a matter of debate. Results from different investigations in animal models or patients with Parkinson's disease vary. Carvey et al.  observedet al. found that P-glycoprotein exhibits decreased function in patients with advanced but not early Parkinson's disease [et al. [in vivo neuroimaging techniques with gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). With regard to BBB transport for L-DOPA being dependent on the L-type amino acid influx transporter 1 (LAT1) [et al. [et al. [et al. [Furthermore, they found areas with increased expression of P-glycoprotein (P-gp), and showed that the dopamine antagonist domperidone, that normally has highly limited brain distribution due to its high affinity for P-gp, was able to attenuate apomorphine-induced stereotypic behaviour in these animals. In human positron emission tomography (PET) studies, Bartels  disease ,13. The  disease . Also, i [et al.  found no1 (LAT1) , it is o [et al.  found a  [et al.  found BB [et al.  found anmutual interactions of BBB transport, the fate of L-DOPA metabolism in the brain, and the number of viable dopaminergic neurons under normal and diseased conditions are needed for improved understanding of Parkinson's disease. In this study using a unilateral rat model of Parkinson's disease, we characterized the BBB transport of L-DOPA, L-DOPA's neuropharmacokinetics and its associated conversion, via dopamine, into the main dopamine metabolites DOPAC and HVA, in healthy as well as in late-stage diseased brain. Our hypothesis was that a decrease in BBB transport of L-DOPA would contribute to the decline in L-DOPA efficacy at later stages of Parkinson's disease.Studies on the potential In this study, intracerebral microdialysis was used for the determination of BBB transport of L-DOPA . At 14 dad libitum.All animal procedures described in this paper were approved by the Ethical Committee on Animal Experimentation of the University of Leiden (DEC numbers 118 and 5069). Experiments were performed on male Lewis rats  weighing 311 ± 17 g  and 288 ± 13 g  before surgery and before start of the experiment, respectively. The rats were housed in standard plastic cages  with a 12-hour day/night schedule (lights on 7:30 a.m.) and at a temperature of 21°C. The animals had access to standard laboratory chow  and acidified water The surgery for the microdialysis study was performed under anesthesia with an intramuscular injection of 0.1 mg/kg medetomidine hydrochloride  and 1 mg/kg ketamine base . Three indwelling cannulae  were implanted, one in the left femoral artery for blood sampling and two in the left femoral vein for drug administration. The cannulae were tunnelled subcutaneously and fixed at the back of the neck with a rubber ring. The skin in the neck was stitched with normal sutures. The skin in the groin was closed with wound clips. To prevent clotting and obstruction, the cannulae were filled with a 25% (w/v) polyvinylpyrrolidone solution  in pyrogen-free physiological saline  containing 20 IU/ml heparin .® Sigma Alldrich BV, Zwijndrecht, the Netherlands) at a rate of 0.1 μl/min for 30 min. Rotenone was dissolved in a 1:1-mixture of dimethylsulfoxide  with polyethylene glycol . After the infusion, the needle was kept in place for another 5 min to prevent leakage along the track of the needle. Subsequently, two small holes were drilled into the skull to allow implantation of the microdialysis guide cannulae  in the left and in the right striatum, AP: +0.4; L: +/-3.2; V: -3.5 relative to bregma [After the implantation of the intravascular cannulae, the rats were placed in a stereotaxic frame and the skull was exposed for brain surgery. The skull was cleaned and a hole was drilled to allow a needle  to be lowered into the right MFB using the co-ordiantes AP: -2.8; L: +2.0; V: -9.0 relative to bregma  for unilo bregma . One supo bregma .2 1.2; MgCl2 1.0; ascorbic acid 0.2 in a 2 mM phosphate buffer pH 7.4) [At 13 days after the unilateral infusion of rotenone into the right MFB, and 18-24 hours prior to the experiment, the microdialysis probes  were inserted into the guide cannulae. All animals were fasted overnight prior to the experiment in order to rule out any competition in BBB transport of L-DOPA with food-related amino acids . The mic pH 7.4)  at a floin vivo recovery of L-DOPA was determined by the retrodialysis method. For this purpose, the probes were first perfused with a L-DOPA solution  for 60 min to collect 6 fractions. The relative loss of L-DOPA was determined and used for estimating brain extracellular fluid (brainECF) concentrations. After this period, the syringes were switched to blank perfusion fluid for the washout phase of 90 min.After a stabilisation period of 60 min, the 3 to neutralize the acidic L-DOPA solution. Both infusions were started at the same time for 20 min at a rate of 20 μl/min. In the first 120 min of the experiment, microdialysis fractions were collected at 10-min intervals. From 120-180 min, microdialysis fractions were collected at 20-min intervals. From 180 min until the end of the experiment (at 360 min), microdialysis fractions were collected at 30-min intervals. Blood samples  were taken at pre-dose (5 min before), and at 5, 10, 15, 20, 22, 24, 26, 28, 30, 45, 50, 60, 75, 90, 120, 180, 240, 360 min after start of the L-DOPA infusion. The blood samples were centrifuged for 10 min at 5000 rpm and the plasma was pipetted into Eppendorf vials. All samples were stored at - 80°C before analysis. After the experiment, the animals were given an overdose of sodium pentobarbital  and the thorax was opened and the vascular bed was perfused via the left ventricle of the heart with 30 ml of saline followed by 30 ml of 10% phosphate buffered formalin (pH 7.0). Brains were removed for histopathology.After the washout period, the intravenous administration of L-DOPA was started. One venous cannula was connected to a syringe containing L-DOPA  in 0.2 M HCl in saline (0.9% NaCl) and ascorbic acid (5% of the L-DOPA amount) and the second venous cannula was connected to a syringe containing 7% NaHCOTH immunohistochemistry was performed to quantify the degree of dopaminergic depletion as described previously . After sAll plasma samples were analysed for L-DOPA and all microdialysate samples were analysed for L-DOPA, dopamine, DOPAC and HVA using a high performance liquid chromatography (HPLC) system with electrochemical detection (ECD).in situ Ag/AgCl (ISAAC) reference electrode operating in the DC mode. For the analysis, a standard Ag/AgCl reference electrode, filled with a saturated KCl solution was used. Data acquisition and processing was performed using the Empower® data-acquisition software (Waters).The HPLC system consisted of a LC-10AD HPLC pump , a Waters 717 Plus autosampler , a pulse damper  and a digital electrochemical amperometric detector . The electrochemical detector consisted of a VT-03 electrochemical flow cell combined with a 25 μm spacer and an ® 5 μm C-18 column  equipped with a refill guard column  packed with pellicular C18 material  at a constant temperature of 30°C. The mobile phase was a mixture of 0.05 M sodium phosphate buffer (pH 2.8) and methanol , supplemented with 0.3 mM EDTA  and 10 mM octane-sulfonic acid. Before the addition of methanol, the mobile phase was filtered through a 0.2 μm nylon filter (Alltech), then the methanol was added and it was mixed and degassed with helium. The flow rate was set at 1 mL/min. The optimal working potential for L-DOPA was +0.75 V, as determined by a voltammogram and sensitivity plot. Concentrations were measured at a sensitivity range of 5 nA for L-DOPA and 20 nA for 3,4-dihydroxybenzylamine hydrobromide . Stock solutions of L-DOPA were prepared at a concentration of 1 mg/mL in Millipore water. The stock solutions were diluted with Millipore water to obtain calibration solutions in the range of 2 to100 ng/mL. The internal standard (DHBA) solution was prepared by dilution of the stock solution to a final concentration of 500 ng/mL. The stock solutions were stored at -80°C for up to one month. The assay solutions were prepared freshly before each analysis. For determination of the L-DOPA in plasma, 25 μL of internal standard solution (DHBA 500 ng/mL) was added to 45 μL plasma samples and 50 μL of Millipore water in glass centrifuge tubes. Next, 25 μL of 20% TCA was added and the mixture was vortexed for 5 min. After centrifugation for 10 min at 4000 rpm (2000 g), 100 μL of the supernatant was added to 50 μL of phosphoric buffer  of which 25 μL was injected into the HPLC system.Chromatography of plasma samples of L-DOPA was performed on a Beckman Coulter™ Ultrasphere® 5 μm C-18 column  at a constant temperature of 30°C. The mobile phase was a mixture of 0.05 M sodium phosphate buffer (pH 2.8) and methanol , supplemented with 0.3 mM EDTA  and 1.5 mM octane-sulfonic acid. Mobile phase solvents were filtered through a 0.2 μm nylon filter. The methanol was added and the mobile phase was mixed and degassed with helium. The flow rate was set at 0.2 mL/min. The optimal working potential for a mixture of L-DOPA, dopamine, DOPAC and HVA was +0.66 V, as determined by a voltammogram and sensitivity plot. Concentrations were measured at a sensitivity range of: 0.1 nA for dopamine, 0.5 nA for L-DOPA, HVA and isoproterenol; and 10 nA for DOPAC. For analysis of brain microdialysate samples, stock solutions of L-DOPA, DOPAC and HVA were prepared at a concentration of 0.5 μg/mL for dopamine, 1 μg/mL for L-DOPA and 5 μg/mL for DOPAC and HVA in microdialysis perfusion fluid with aqueous antioxidant solution consisting of 0.1 M acetic acid, 3.3 mM L-cysteine, 0.27 M EDTA  and 0.0125 mM ascorbic acid (4:1 v/v). Internal standard solution was freshly prepared before each analysis by dilution of a 1 mg/mL isoproterenol stock solution, to a concentration of 100 ng/mL of the compound in perfusion fluid that contains antioxidant (4:1 v/v). All the stock solutions were stored at -80°C for up to one month. Before each analysis a first calibration solution containing all compounds was freshly prepared by mixing one volume of each stock solution  and adding 5 volumes of perfusion fluid with antioxidant (6:1 v/v). This first calibration solution now contained all compounds at a concentration 10 times lower than their stock solution and from this solution the other calibration solutions were prepared.For analysis of L-DOPA, dopamine, DOPAC and HVA brain microdialysate concentrations, 5 μL of internal standard  solution was added per 10 μL of microdialysis sample or calibration curve sample. The samples were then injected (20 μL) into the HPLC system without further sample pre-treatment. Chromatography of brain microdialysate samples was performed on a Beckman Coulter™ UltrasphereThe pharmacokinetics of L-DOPA, DOPAC and HVA were analysed utilizing a population pharmacokinetic modelling approach. Dopamine concentrations were all below the limit of detection. Compartmental modelling was performed using the ADVAN6 subroutine in NONMEM VI release 2 . All fitting procedures were performed on an IBM-compatible computer  running under Windows XP with the Compaq Visual Fortran compiler version 6.6. The inter-individual variability of model parameters was described by an exponential equation, according to:1 is the population  estimate for parameter P1, P1i is the individual estimate and ηi determines the random deviation of P1i from P1. The values of ηi are assumed to be randomly, normally distributed with mean zero and variance ω112. The residual error in the L-DOPA concentration was described by a proportional error model:where θand the residual error in the DOPAC or HVA concentration was described by an additive error model:obs, ij represents the jth measured L-DOPA, DOPAC or HVA concentration for the ith individual predicted by the model. Cpred, ij represents the prediction of concentration and εij is the deviation of the model-predicted value from the observed concentration. The values of ε are assumed to be randomly, normally distributed with mean zero and variance σ2.where C2's and σ2's. Structural model selection for all models was based on the likelihood ratio test, diagnostic plots , parameter correlations and precision in parameter estimates. Inclusion of one parameter into the model was assumed to be significant if this led to a decrease of 10.8 points or more of the minimum value of the objective function (MVOF) after fitting the model to the data. This corresponds to a theoretical significance level of p = 0.001 under the assumption that the difference in MVOF between two nested models is χ2 distributed.The first order conditional estimation method with interaction (FOCE interaction) was used in NONMEM to fit the models to the data and to estimate θ's, ωECF profiles from the control cerebral hemisphere of 12 rats  and the L-DOPA brainECF profiles from the rotenone-treated responder cerebral hemisphere of 7 responder rats  were included in the population pharmacokinetic analysis.In total, the L-DOPA plasma profiles of 13 rats , the L-DOPA brainECF L-DOPA data from the individual rats were simultaneously analysed  for each rat from this analysis and typical values from this model  in order to estimate brainECF concentrations. The in vivo recovery was equal for the three concentrations of L-DOPA used, and not affected by disease condition.The percentage of intact TH staining in the rotenone-treated cerebral hemisphere compared to the untreated hemisphere at the level of the striatum was below 40% in 12 out of 17 rats (responders), and higher than 90% in the remaining 5 rats (non-responders). No quantifiable concentrations of dopamine could be detected throughout the experiment . A total of 17 rats were used in the microdialysis experiments: n = 6 in the 10- and 25 mg/kg dose group and n = 5 in the 50 mg/kg dose group, where for one rat the microdialysis probe malfunctioned. All microdialysate concentrations of L-DOPA were corrected for the average ECF concentration-time profiles of L-DOPA are shown in Figure ECF were analysed simultaneously. All structural parameters of the population pharmacokinetic model for L-DOPA could be adequately estimated (Table in) for compartment 4 and 5 resulted in similar estimated values (5.8 min-1). Striatal brainECF baseline L-DOPA levels in the rotenone-treated responder cerebral hemisphere were not statistically different  from those in the control hemisphere and averaged 0.010 ± 0.004 pmol/mL and 0.024 ± 0.011 pmol/mL (mean ± SEM), respectively. Therefore, it can be concluded that no significant difference can be identified between the pharmacokinetics of L-DOPA in the control versus the rotenone-treated responder cerebral hemisphere.The plasma concentration-time profiles for each rat following intravenous infusion of 10, 25, and 50 mg/kg of L-DOPA are shown in Figure p-value < 0.01; Welch's t-test). Also, striatal microdialysate baseline HVA dialysate levels in the rotenone-treated responder cerebral hemisphere were lower (approximately 4 times) than in the control cerebral hemisphere with respective values of 0.25 ± 0.14 pmol/mL and 0.9 ± 0.08 pmol/mL (mean ± SEM), respectively . Figure Striatal microdialysate baseline DOPAC levels in the rotenone-treated responder cerebral hemisphere were about 6 times lower than in the control cerebral hemisphere and averaged 0.2 ± 0.19 pmol/mL and 1.3 ± 0.17 pmol/mL (mean ± SEM), respectively (ECF pharmacokinetics (BBB transport) of L-DOPA and its conversion into the dopamine metabolites DOPAC and HVA, was measured in parallel in the rotenone-treated responder and control cerebral hemisphere at 14 days post-rotenone injection. NONMEM was used to develop a population based pharmacokinetic model. Basal concentrations of DOPAC and HVA in striatal microdialysate were lower in the rotenone-treated responder brain than in the control brain. Furthermore, it was shown that disease-related changes were observed in the kinetics of the dopamine metabolites following L-DOPA administration, without any changes in BBB transport of L-DOPA.In this study using the rotenone-treated rat as a model for Parkinson's disease, the relationship between plasma and brainIn our study, following the rotenone infusion in the MFB of the rats, the Parkinson's disease state was defined as TH immunostaining in the striatum with a density of less than 40% of control values (termed responders). Data indicated that the number of responders in this model was similar to our previous study , and theECF pharmacokinetics of L-DOPA in control and rotenone-treated conditions are described by a population pharmacokinetic model. The pharmacokinetics of L-DOPA in plasma following intravenous administration was best described by a three-compartmental model, like previously reported [et al. [et al. [The results described in this paper are the first in which both plasma and brainreported . In our  [et al.  found in [et al.  was 3.1 et al. [No difference in BBB transport of L-DOPA between the control and rotenone-treated responder cerebral hemisphere were found. This is in contrast to the findings of an inverse relationship between the MPTP-monkeys' ability to transport L-DOPA from blood to brain and their degree of Parkinsonism by Alexander et al. . This ma-1). Since dopamine-producing neurons are diminished in Parkinson's disease [Also, our results did not indicate a disease-induced change in the endogenous production of L-DOPA in the brain  was indistinguishable from that of the rotenone-treated responder brain (0.000054 min-1). Also, the metabolism of L-DOPA via dopamine to HVA in the untreated brain (0.000023 min-1) was similar to that in the rotenone-treated responder cerebral hemisphere (0.000016 min-1). Furthermore, these values reflect a slow conversion of L-DOPA-via dopamine- to these metabolites. Overall, in contrast to our expectations, it can be said that the disease conditions at 2 weeks post-rotenone-injection into the MFB did not result in any change in the pharmacokinetics of L-DOPA.The main metabolite of L-DOPA in the early phase after drug administration is dopamine ,30, whicThe baseline concentrations of DOPAC and HVA were decreased about 10-fold in the rotenone-treated responder brain Figure . These f-1) was increased about 7-fold compared with the elimination rate constant in the control side (0.0053 min-1). For HVA this disease-induced increase was about a factor 2.5 . With unchanged values for the formation rate constants  in certain renal and hepatic diseases , to our in vivo assessment of dopamine system functionality at different stages of the disease may help to unravel more mechanistically the factors that play a role in effective Parkinson's disease treatment.In general, the integrated approach used in this study, including plasma pharmacokinetics, BBB transport and The developed population pharmacokinetic model allowed the integration of the kinetics of L-DOPA and its conversion to DOPAC and HVA in both control and rotenone-treated responder brain. It was demonstrated that 2 weeks following a unilateral infusion of rotenone into the rat brain, dopamine depletion resulted in lower brain levels and higher elimination rates of DOPAC and HVA for the diseased cerebral hemisphere. This was, however, not accompanied by changes in the plasma pharmacokinetics and BBB transport of L-DOPA. Taken together, the varying results on whether or not changes in BBB transport in Parkinson's disease in previous studies and in this study indicate that changes in BBB functionality are not specifically associated with Parkinson's disease, and therefore cannot account for the decreased benefit of L-DOPA at later stages of Parkinson's disease.BBB: blood-brain barrier; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; L-DOPA: 3,4-dihydroxy-L-phenylalanine; TH: tyrosine hydroxylase; MFB: medial forebrain bundle.The authors declare that they have no competing interests.PR carried out the experiments, modelling of the data and contributed to writing of the manuscript, HD contributed to the model development and writing of the manuscript, MO contributed to the staining of the brain tissues, interpretation of the data and writing of the manuscript, MD participated in the design of the study and revision of the manuscript, EL conceived the project of which this study was a part of, participated in design and coordination of the study, and contributed to writing of the manuscript. All authors have read and approved the final version of the manuscript.


Angiogenesis, the development of new vessels from existing vasculature, plays a central role in tumor growth, survival, and progression. On the molecular level it is controlled by a number of pro- and anti-angiogenic cytokines, among which the vascular endothelial growth factors (VEGFs), together with their related VEGF-receptors, have an exceptional position. Therefore, the blockade of VEGF signaling in order to inhibit angiogenesis was deemed an attractive approach for cancer therapy and drugs interfering with the VEGF-ligands, the VEGF receptors, and the intracellular VEGF-mediated signal transduction were developed. Although promising in pre-clinical trials, VEGF-inhibition proved to be problematic in the clinical context. One major drawback was the generally high variability in patient response to anti-angiogenic drugs and the rapid development of therapy resistance, so that, in total, only moderate effects on progression-free and overall survival were observed. Biomarkers predicting the response to VEGF-inhibition might attenuate this problem and help to further individualize drug and dosage determination. Although up to now no definitive biomarker has been identified for this purpose, several candidates are currently under investigation. This review aims to give an overview of the recent developments in this field, focusing on the most prevalent tumor species. The growth and progression of tumors is crucially dependent on the supply of oxygen and the exchange of nutrients and metabolites with the surrounding tissue. As transport based on diffusion of these molecules is limited to very short distances of less than 3–4 mm only, a tumor exceeding this size needs to develop a vascular system in order to survive. The most important process driving this neovascularization is angiogenesis, the development of new vessels from the existing vascular system ,2. PhysiOn the molecular level, angiogenesis is controlled predominantly by the relatively small family of vascular endothelial growth factors (VEGFs). It comprises VEGF-A (commonly named VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta growth factor (PlGF) –12. MoreThe VEGF proteins are ligands for three tyrosine kinase receptors: VEGF receptor 1 , VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) –28. VascVEGF-A is the central factor in the promotion and regulation of tumor angiogenesis, VEGF signaling is mainly mediated by VEGFR-2 ,34, whilConsidering the outstanding importance of angiogenesis for tumor growth and survival in general and the role of the VEGF-A/VEGFR-2 signaling system in particular, several drugs have been developed, which interfere with different angiogenic molecules. In principle, three different modes of action are possible: interception of the VEGF ligand, blockade of the VEGFR, or interruption of the intracellular VEGFR-mediated signaling.Examples for drugs targeting the VEGF-A ligand are Bevacizumab and Aflibercept. Bevacizumab is a VEGF-binding humanized recombinant antibody, which inhibits the VEGF-VEGFR-interaction . In the Ramucirumab, a human antibody specific for the extracellular ligand-binding domain of VEGFR-2, belongs to the class of VEGFR-blocking drugs . RamucirSmall-molecule tyrosine kinase inhibitors (TKIs) inhibit ATP binding to the tyrosine domain of VEGFRs and therefore interrupt the VEGFR signal transduction. The most prominent members of this class of drugs are Sunitinib and Sorafenib, which are primarily used in renal  and gastin vitro experiments, our group could demonstrate that cellular changes [However, although many studies seem to indicate a modest benefit of anti-angiogenic therapy, it generally suffers from a high variability in the response by the individual patient. Possible reasons for this observation may be the general capability of endothelial cells to form new vessels independently of an enhancement of the VEGF-A/VEGFR-2 signaling system. In a series of  changes ,83 induc changes ,84,85. TCancer cells are usually classified by biomarkers. A biomarker, as defined by the NIH, is “a characteristic objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” . SeveralColorectal cancer is the second most frequent cause of death in North America and Europe with about 600,000 deaths and a rate of approximately 1.2 million new cases per year worldwide . At the et al. [et al. [Blood is a relatively easily available material for the analysis of biomarkers and has a great potential for this application. Several studies have analyzed circulating molecules for their potential as predictive biomarkers for colorectal cancer. Cetin et al.  found th [et al.  have scrAngiopoietin-2 is another potentially valuable circulating biomarker. It has been proposed to be involved in VEGF function and vascular remodeling. Interestingly, low serum angiopoietin-2-levels were found to be associated with a high overall survival (OS) of >90% after 18 months and a better response rate to anti-angiogenic therapy of >80% compared to high serum angiopoietin-2-levels in a study analyzing patients receiving Bevacizumab in combination with different chemotherapeutic regimens  .Finally, circulating endothelial cells (CEC) have been found to predict the response to Bevacizumab-therapy in colorectal cancer. Blood from patients receiving FOLFOX4 and Bevacizumab was analyzed for endothelial cells before and during treatment. It could be shown that no correlation existed between the CEC levels and the outcome in a FOLFOX4 alone control group. However, CEC proved to be a strong indicator for the outcome of the Bevacizumab-based therapy. Patients with less than 65 CEC/4 mL blood at baseline (as determined by the CellSearch system), had a significantly longer median PFS and OS than patients with 65 CEC/4 mL or more. In addition, a low proportion of CXCR4-positive CEC of below 20% at baseline was also correlated to significantly longer PFS and OS ,96. ThesVEGF, KDR, IL6, CXR1 and -2, P53, MMP2,-7,-9, and ICAM genes of patients with metastatic colorectal cancer receiving FOLFOX or XELOX with Bevacizumab using genomic DNA from peripheral blood. The IL-6 G-174C and P53 codon 72 polymorphisms were found to be correlated to a positive response to Bevacizumab therapy. Furthermore, PFS was significantly associated with MMP9 C-1562T and CXCR-1 G + 2607C [Genes involved in angiogenesis show a relatively high level of variation, ranging from silent SNPs to functional polymorphisms. The latter were tested in the  + 2607C .vs. CA = 40% vs. AA = 56%, p = 0.048, or CC + CA = 39% vs. AA = 56%, p = 0.015), indicating that the A-allele exerts a strong beneficial effect [In another study, the Nordic ACT trial , the VEGFR-1 319 C/A SNP has been identified as significantly associated with response to Bevacizumab. Objective response rates differed significantly between the three genotypes  after treatment with FOLFOX/Bevacizumab as compared to the rest.CD133, a trans-membrane protein isolated from colorectal cancer stem cells ,102, haset al. concluded in their recent paper that early treatment-related blood pressure increases do not predict clinical benefit from Bevacizumab based on PFS or OS outcomes. BP increases do not appear to have general prognostic importance for patients with advanced cancer [et al. observed that Bevacizumab-induced grade 2–3 hypertension in patients receiving Irinotecan, Fluorouracil, and Bevacizumab was significantly associated with improved PFS  [et al. described that hypertension was associated with prolonged PFS  and OS  in patients treated with Bevacizumab-containing chemotherapy [et al. observed an induced grade 2–4 hypertension in 17.6% of the cases. Of the patients with induced arterial hypertension, 84.6% achieved a complete or partial response, whereas patients without these side effects only had 41.9% (p = 0.006). In addition and comparable to the other studies, hypertension was associated with improved PFS  [Hypertension is a very common side effect of VEGF inhibitor medications, with general incidence rates of about 20% and grade 3 hypertension percentages of approximately 11% , 104. Hud cancer . This id = 0.04) . Österluotherapy . Develop = 0.04) .Among women, breast cancer is the malignancy with the highest occurrence at a rate of about 23% and a total number of approximately 1.4 million new cases per year worldwide. Furthermore, with about 460,000 cases per year it is also the most frequent cause of death due to cancer . Unfortuet al. could demonstrate that baseline VEGF plasma levels were associated with the time to progression (TTP). Patients with VEGF concentrations >32.6 pg/mL had a median TTP of 3.7 months, whereas patients with VEGF levels <32.6 pg/mL had a median TTP of 9.3 months (p = 0.003) [p = 0.033 and p = 0.035, respectively) predictors of clinical response in the form of operability [et al. measured the VEGF, soluble VEGFR-2 (sVEGFR-2), soluble VEGFR-3 (sVEGFR-3), and soluble KIT (sKIT) plasma levels at baseline and during therapy in patients receiving Sunitinib after chemotherapy with and Anthracycline or Taxane. They found a trend for a connection of decreasing sVEGFR-3 levels (≥20%) with longer OS (p = 0.07). In addition, decreases of sKIT levels by ≥50% were significantly associated with longer TTP (p < 0.001) and OS (p = 0.0194) [i.e., repeated, low-dosed, chemotherapy in conjunction with Bevacizumab. A significant association of high baseline levels of CEC with overall response (p = 0.02), clinical benefit (p = 0.01), and improved PFS (p = 0.04) was observed in this study [In a study with combined Bevacizumab/Vinorelbine chemotherapy Burstein = 0.003) . In addirability . Burstei 0.0194) . Finallyis study .et al. did a retrospective study on the ECOG2100 cohort, investigating five SNPs for VEGF and two for VEGFR2. They found that VEGFR2 polymorphisms did not show any influence on OS or any other clinical parameter. Two VEGF genotypes, on the other hand, VEGF-2578 AA and VEGF-1154 AA, were significantly associated with improved OS  in the Paclitaxel/Bevacizumab combination arm of the study [So far, only polymorphisms in the VEGF and VEGFR2 genes have been analyzed for their potential to serve as a biomarker for anti-angiogenic therapy in breast cancer. Schneider he study .et al. also observed in the same study that patients who developed grade 3–4 hypertension had an improved median OS of 38.7 months vs. 25.3 months of normotensive patients (p = 0.002) [vs. Bevacizumab + placebo) did not show a prognostic value of early hypertension [The role of anti-angiogenic therapy induced hypertension is subject of debate, as currently there are contradicting results found in the literature. Beside identifying two VEGF-SNPs as candidate biomarkers, Schneider = 0.002) . In contrtension . FurtherBlood levels of VEGF and related molecules can be influenced by many different additional factors besides cancer and may therefore be misleading or difficult to interpret in an analysis of their predictive value. Hence it might be a better approach to directly determine the expression of candidate genes or proteins inside the tumor tissue itself. This, of course, poses some problems, like easy availability of ideally multiple samples over the course of the therapy.et al. immunohistochemically investigated a panel of biomarker candidate proteins in patients receiving a first line Paclitaxel/Bevacizumab therapy against metastatic breast cancer. They found that high intra-tumoral expression of VEGFR-3 was associated with clinical response, whereas VEGFR1 overexpression was an indicator for poor survival [Fountzilas survival .et al. demonstrated that MMP2, PTTG, VEGF-C, CXCR4 and bFGF are potential cellular tumor markers for identifying thyroid cancer with greater risk for metastasis. The authors suggest that the combination of the angiogenic factors VEGF-C and bFGF favors progression in metastatic thyroid carcinoma [Although representing only a small portion of cancer cases with an incidence of about 213,000 new cases (which equates to approximately 1.7%) and a mortality of a bit over 35,000 cases per year worldwide, thyroid cancer is the most frequent malignant endocrine tumor ,117. Forarcinoma . Thus, tarcinoma . Subsequarcinoma –133. Theet al. showed in 2011 [Broutin  in 2011  that cyt in 2011 . In addi in 2011 . Therefoet al. analyzed a cohort of patients with progressive advanced thyroid cancer receiving Motesanib as anti-angiogenic therapy over a period of 48 weeks [p = 0.0007). Furthermore, the study could show that not only absolute serum concentrations or activities but also their changes during treatment were a useful indicator for patient response to therapy. Increases of PlGF by more than 4.7 fold (p < 0.0001) and of caspase 3/7-activity by more than 2.1-fold (p < 0.0001) as well as changes by less than −1.6-fold of sVEGFR2 (p < 0.0001) were independent predictors enabling investigators to separate responders from non-responders [Bass 48 weeks . During sponders . In anotsponders . Furthersponders . Now, sesponders  or as a sponders ,141.et al. demonstrated that hypoxia-inducible factor-1alpha is expressed in papillary thyroid carcinomas and is not only regulated by hypoxia but also by the BRAF(V600E)-mediated signaling pathway [Shaik and coauthors described a subtype of human papillary thyroid cancer (PTC), which is resistant to therapy with VEGF receptor 2 (VEGFR2)-inhibitor. In these poorly differentiated PTC cells, the beta-transducin-repeat-containing protein inhibits cell migration and decreases sensitivity to Sorafenib . In vari pathway . BRAF has been tested as predictive markers of anti-angiogenic treatment in phase III Treatment Approaches in Renal Cancer Global Evaluation Trial (TARGET). In this study high baseline levels of VEGF were associated with poor prognosis but baseline sVEGFR-2 and changes in VEGF or sVEGFR-2 could not predict the response to Sorafenib . In phaset al. were able to identify a candidate CAF signature that may help predict PFS benefit from Sorafenib treatment in patients suffering from metastatic RCC [High levels of CAIX protein was shown to correlate to the responsiveness of interleukin (IL)-2 with 78% of patients responding to IL-2 showing high expression (>85% tumor cells) of CAIX protein in tumors compared to 51% in non-responders. The value of CAIX as a predictive marker is currently under investigation ,157. Simatic RCC ,163.Genome-wide association studies have reported SNPs that may increase the risk of developing RCC ,165. A lAmong men prostate cancer (PC) is the second most frequently occurring cancer with about 900,000 new cases and approximately 260,000 deaths per year worldwide. It also has by far the highest worldwide 5-year prevalence of roughly 24% . In USA So far, most analyses of possible biomarker candidates for the prognosis and prediction of anti-angiogenic therapy have been conducted with a rational approach , concentAnti-angiogenic therapy generally suffers from a high variability in the response by the individual patient. In order to select patients with the highest likelihood for a positive response to such a treatment, the availability of reliable predictive biomarkers for anti-angiogenic therapy will be a key factor. Although there are some promising preliminary results, no general or cancer-specific biomarker has yet emerged, which could help select patients with a positive prognosis for anti-angiogenic therapy. For its future it is therefore of vital importance to conduct larger systematic trials to translate the preclinical data into clinically usable systems and to switch from unselective therapy to a more individual drug selection based on the patients’ predispositions.


Stratum 4 covers the distal half of the human X chromosome short arm and the p-distance of the stratum is ~10%, on average. However, a 100-kb region, which includes KALX and VCX, in the middle of stratum 4 shows a significantly lower p-distance (1-5%), suggesting frequent sequence exchanges or gene conversions between the X and Y chromosomes in humans. To examine the evolutionary mechanism for this low p-distance region, sequences of a corresponding region including KALX/Y from seven species of non-human primates were analyzed.Mammalian sex-chromosomes originated from a pair of autosomes. A step-wise cessation of recombination is necessary for the proper maintenance of sex-determination and, consequently, generates a four strata structure on the X chromosome. Each stratum shows a specific per-site nucleotide sequence difference . A KALY-converted KALX allele in white-handed gibbons also suggests a possible recent gene conversion between the X and Y chromosomes. In these primate sequences, the proximal boundary of this low p-distance region is located in a LINE element shared between the X and Y chromosomes, suggesting the involvement of this element in frequent gene conversions. Together with a palindrome on the Y chromosome, a segmental palindrome structure on the X chromosome at the distal boundary near VCX, in humans and chimpanzees, may mediate frequent sequence exchanges between X and Y chromosomes.Phylogenetic analysis of this low LINE element at the proximal end of the region may be a cause for these frequent conversions. This gene conversion in humans may also be one of the genetic causes of Kallmann syndrome.Gene conversion events between the X and Y homologous regions have been suggested, mainly in humans. Here, we found frequent gene conversions in the evolutionary course of primates. An insertion of a  SRY (sex-determining region on Y) and RBMY , on one of the proto-sex chromosomes, the proto-Y chromosome. The tight linkage of sex-determining genes on the proto-Y chromosome is required for normal development of male characters or male determination [The mammalian sex chromosomes (X and Y chromosomes) differentiated from a pair of autosomes (proto-sex chromosomes) more than 200 million years (myr) ago, before the divergence of monotremes from other mammals ,2. The pmination , and themination ,5.KS) for 19 X-Y pairs of genes in humans or squirrel monkeys varied greatly from 0.05 at ARSE/ARSEP to 1.25 at SRY/SOX3. In addition, the KS of the 19 pairs decreased with position on the X chromosome from the long arm tip to the short arm tip. Based on this variation, the 19 pairs were classified into four distinct categories, or strata. Stratum 1 contains three genes with KS > 0.9, stratum 2 contains two genes with KS of approximately 0.5, and strata 3 and 4 comprise seven genes with KS = ~0.2, and KS = ~0.1, respectively. The KS values in strata 1 and 2 suggest that recombination between X and Y chromosomes ceased before the divergence of monotremes from other mammals for stratum 1, and before the divergence of marsupials and eutherian mammals for stratum 2. The observed KS values of ~0.2 and ~0.1 also suggest that stratum 3 formed before the mammalian divergence and that stratum 4 formed in the primate lineage after the divergence of prosimian and simian primates. To confirm whether this step-wise divergence between X and Y chromosomes is observed in other non-coding regions on the X and Y chromosomes, Iwase et al. [p-distances and confirmed the scenario of sex chromosome differentiation proposed by Lahn and Page [p-distances within stratum 4, where the p-distance is ~0.1 on average, were significantly lower (0.01~0.05)  neurons and olfactory neurons to the hypothalamus [KALX, KALY, is located on the long arm of the Y chromosome, but because of deletions and nonsense mutations the gene is a pseudogene [KALX and KALY are in the middle of stratum 4, the two genes began to differentiate after simian primates diverged from prosimians [syndrome -12, is lsyndrome ,13,14. Asyndrome  suggestithalamus -21. A hoeudogene ,23. Sincosimians .VCX and VCY form a relatively small gene family, with four members of VCX and two of VCY being identified in humans. Each member of the VCX gene family has three or four exons and is 1320 ~1880 bp in length, whereas VCY genes have two exons and are 742 bp long. All members of VCX and VCY are expressed exclusively in male germ cells [VCX and VCY sequences show a high degree of sequence similarity within each gene family as well as between genes on the X and Y chromosomes [VCY is due to frequent gene conversion between palindrome arms [rm cells . VCX andomosomes . Sequencome arms ,27.p-distance region within stratum 4 in non-human primates. To this end, we compared nucleotide sequences of corresponding regions between X and Y chromosomes of several primates. The analysis showed that relatively frequent gene conversions between the X and Y chromosomes have occurred independently in each of the primate lineages. This frequent gene conversion is argued to be a result of genomic structure, such as repetitive elements at the boundary of the region.The purpose of this study was to examine the evolutionary mechanism that produces the low p-distances between X and Y chromosomes in chimpanzees shows a low p-distance region on the X chromosome that corresponds to a similar low p-distance region on the X-chromosome in humans. With the exception of the centromeric region containing KALX or KALY , whereas the corresponding regions in humans have values of p = 0.01 and 0.05, respectively  and no further exchanges occurred. Therefore, sequences from the three primates show the reciprocal monophyly of KALX and KALY. In contrast, Region B shows rather species-specific clustering, suggesting independent gene conversion in these species.The formation of a low d) on the branches leading to the X and Y chromosome sequences in Regions A and B were calculated. Since the nucleotide substitution rates of the X and Y chromosomal genes differ [d values on the X and Y chromosomes are estimated separately on the tree  and KALY (dY) in rhesus monkeys and hominoids from a common ancestor of these species is given by dX = 0.023 ± 0.007 and dY = 0.041 ± 0.006 for Region A, and dX = 0.023 ± 0.003 and dY = 0.046 ± 0.001 for Region B. The values of dX and dY in Region A are similar to those in Region B. This is consistent with the gene conversion that does not affect species divergence. The divergence re-estimated using both sequences in Regions A and B gives dX = 0.023 ± 0.005 and dY = 0.044 ± 0.005. Assuming a divergence time between rhesus monkeys and hominoids of 30 myr [KALX and KALY is 0.78 ± 0.18 × 10-9 and 1.45 ± 0.17 × 10-9 per site per year, respectively. Based on these substitution rates, the timing of gene conversion in Region B for rhesus monkey X and Y chromosomes was estimated to be 17~28 myr ago and that for hominoids was 19~24 myr ago  staining of the chromosomes. A single copy of KAL was detected on the X and Y chromosomes of each species, with the exception of lemurs  KALX and KALY, reflecting an ancient gene conversion between X and Y in the stem lineage of Catarrhini.To test whether gene conversion can be observed in other primates, specifically gorillas, gibbons, New World monkeys, and ring-tailed lemurs, the chromosomal positions of To understand whether or not there are hot spots of conversion in the B region, we analyzed the 7 kb region in detail. We investigated a total of 20 NJ trees with a sliding window width of 500 bp and an overlap of 100 bp for the B region. Based on the topologies of the 20 trees, the B region was further classified into seven sub-regions, each of which shows signatures of gene conversion Figure . Region KALX and KALY have occurred during primate evolution. The direction of gene conversion was inferred from the phylogenetic analysis. Among the conversions, five were from KALY to KALX and the remaining five were in the opposite direction. We also identified an example of ongoing gene conversion from Y to X chromosomes in white-handed gibbons. The alignment of four KALX and two KALY sequences from four  white-handed gibbons  is identical to a KALY sequence (white-handed gibbon 2846 MY). Since these KALX and KALY sequences differ by eight substitutions, it is unlikely that all eight substitutions are the result of recurrent substitutions on a particular region of the KALX allele sequence. This observation is likely an example of a recent gene conversion in this region in a non-human primate.Numerous (at least ten) independent inter-chromosomal gene conversions between s Figure  indicateLINE, SINE, and simple repeats, which are known to be involved in sequence exchanges, were searched for at the boundary of the low p-distance region of humans, chimpanzees, and rhesus monkeys. LINE-1 (L1) retrotransposons represent at least 17% of the human genome [p-distance region does not differ from the average over the entire lengths of the X and Y chromosomes. KALX and KALY, however, share L1 elements (about 3 kb) near the boundary , although no one-to-one orthologous relationship with the four human VCX sequences was detected, suggesting species-specific gene duplications or frequent gene conversions within the VCX genes on the X chromosome in New world monkey.The low mpanzees . In contGene conversion is an event in genetic recombination that occurs at high frequencies not only in germ cells through meiotic division but also in somatic cells through mitosis. Recombination occurs between molecules possessing similar nucleotide sequences and is a common repair mechanism for nucleotide lesions in both prokaryotes and eukaryotes. Recombination is initiated by a double-strand break in one gene followed by the repair of this gene by copying the sequence of a similar gene. In the process of this repair, a "Holliday structure" is constructed and depending on the resolution of this structure, the result is called either gene conversion or crossing over ,32. HoweL1 element may play an important role in the repair process. Although the mechanism is not yet well defined, double-strand breaks at chromosomal LINE elements can be repaired by gene conversion with another LINE element in cis or trans [L1 elements that have resulted in an approximately 0.5 Mb reduction in the amount of human DNA within the past few million years.An or trans . Such coor trans ,35. In for trans  identifiLINE element at the boundary of the low p-distance region and found that the boundary was located within this LINE element , gorilla (Gorilla gorilla), agile gibbon (Hylobates agilis), white-handed gibbon (Hylobates lar), rhesus monkey (Macaca mulatta), cotton-top tamarin (Saguinus oedipus), squirrel monkey (Saimiri sciureus), and ring-tailed lemur (Lemur catta). The sequenced regions and accession numbers are listed in Additional file Genomic DNA samples were provided from Kyoto University or Max Planck Institute for Biology. These DNA samples were of the following species: chimpanzee . Amplifications were carried out using a RoboCycler Gradient 96 instrument (Stratagene) with the following standard conditions: denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 sec, 56-62°C for 30 sec and 72°C for 6 min, and a final extension at 72°C for 10 min. For rhesus monkeys, we used LA ExTaq DNA polymerase (TaKaRa) to amplify long fragments (>10 kb) with PCR conditions of denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 56-62°C for 1 min and 69°C for 6 min, and a final extension at 69°C for 10 min. These PCR conditions were modified slightly for some primer sets.PCR was performed in reaction volumes of 20 μl using 20 pmol of each primer, 100 ng of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgClAll PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN). Some products were directly sequenced while others were cloned into pCR-KL-TOPO using a TOPO XL Cloning kit (Invitrogen). Plasmid DNA was purified with a QIAprep Spin Miniprep column (QIAGEN) and used as the template for sequencing reactions. We used the GeneJumper Primer Insertion Kit for Sequencing (Invitrogen) the long rhesus monkey gene sequences.Sequencing reactions were performed using an ABI Prism BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and analyzed on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Sequences for each sample were confirmed by means of independent PCR and subsequent sequencing. To avoid sequencing errors, PCR products or plasmid DNA fragments were sequenced at least twice in both directions. The sequence fragments were assembled by DNASIS (Hitachi) and deposited in the DNA Data Bank of Japan (DDBJ).KALX and KALY from chimpanzees, gorillas, agile gibbons, white-handed gibbons, squirrel monkeys, and ring-tailed lemurs. For rhesus monkeys, we obtained ~50 kb fragments containing exons 5 to 14 of KALX and KALY. These rhesus monkey sequences were used for phylogenetic analysis, together with human and chimpanzee genomic orthologous sequences from GenBank. We detected homologous fragments using DOTTER [We obtained 3 kb exon 5 to 6) and 6 kb (exon 10 to 14) genomic sequences of g DOTTER , made ang DOTTER , and adj to 6 andp-distances (the number of nucleotide differences per site) for all pairwise comparisons of primate sequences. To perform the sliding window analyses of p-distances for these sequences, we used our own FORTRAN program (available upon request). We constructed phylogenetic trees using the neighbor-joining (NJ) method [p-distances in the complete-deletion option and the reliability of the tree was assessed by bootstrap test with 1,000 replications.We computed ) method  and/or t) method  in MEGA4) method . The NJ KALX and KALY are known in humans [KALX and KALY sequences. Slide preparations of the chromosomes for chimpanzees were made from phytohaemagglutinin-stimulated peripheral white blood cells. Chromosome preparations from gibbons, squirrel monkeys and ring-tailed lemurs were made with fibroblasts [KAL sequences for each primate as probes. These probes were labeled using a BioNick labeling kit containing biotin-14-dATP (Gibco BRL) [The chromosomal locations of n humans . To map roblasts . After ibco BRL) . Fluoresbco BRL) . The FISd): per-site nucleotide divergence; (p): per-site nucleotide sequence differences; (KALX): Kallmann syndrome 1 sequence; (KALY): Kallmann syndrome sequence pseudogene; (VCX): variable charge, X-linked; (VCY): variable charge, Y-linked; (ZFX): zinc finger protein, X-linked; (ZFY): zinc finger protein, Y-linked; (PRKX): protein kinase, X-linked; (PRKY): protein kinase, Y-linked along the X chromosome. The position of the KALX and VCX exons are indicated at the bottom of each graph. Each bar corresponds to a p-distance in a non-overlapping window of 500 bp. A low p-distance region is indicated by the two-headed arrows. The region indicated by the rectangle is enlarged in Figure 1B.Click here for fileKALX and KALY probesFISH analysis using species-specific . FISH was carried out on metaphase chromosomes from human , chimpanzee , and rhesus monkey . The X  and Y  chromosomes are marked (arrowheads). Green fluorescence indicates a positive signal. A short white bar marks the centromeric region .Click here for fileKALX and KALY in several primatesThe phylogenetic tree of region A and B of . The trees for Regions A and B are based on p-distances. The numbers of sites are given at the bottom right of each tree. A yellow diamond at the node indicates KALX and KALY differentiation, and the number beside each node is the bootstrap probability after 500 replications.Click here for fileVCX and VCY in several primatesThe phylogenetic tree of . Diamonds indicate gene conversion events.Click here for fileSources of DNA samples and GenBank accession numbers of the analyzed sequences. All the nucleotide sequences reported in this study were deposited in DDBJ (DNA Data Bank of JAPAN) and their accession numbers are AB233497 - AB233526.Click here for filePrimer sets for PCR.Click here for file


It provokes dose-dependent and centrally-mediated sympatholysis. Coronary artery bypass grafting (CABG) with extracorporeal circulation is a stressful procedure increasing sympathetic nervous system activity which could attenuate renal function due the interrelation of sympathetic nervous system, hemodynamics and renal function. We tested the hypothesis that dexmetomidine would improve kidney function in patients undergoing elective CABG during the first two postoperative days.Dexmedetomidine, an alphaThis was a double-blind, randomized, parallel-group study. Patients with normal renal function and scheduled for elective CABG were randomized to placebo or to infusion of dexmedetomidine to achieve a pseudo steady-state plasma concentration of 0.60 ng/ml. The infusion was started after anesthesia induction and continued until 4 h after surgery. The primary endpoint was creatinine clearance. Other variables included urinary creatinine and output, fractional sodium and potassium excretion, urinary potassium, sodium and glucose, serum and urinary osmolality and plasma catecholamine concentrations. The data were analyzed with repeated-measures ANOVA or Cochran-Mantel-Haenszel test.Sixty-six of 87 randomized patients were evaluable for analysis. No significant between-group differences were recorded for any indices of renal function except for a mean 74% increase in urinary output with dexmedetomidine in the first 4 h after insertion of a urinary catheter (p < 0.001). Confidence interval examination revealed that the sample size was large enough for the no-difference statement for creatinine clearance.Use of intravenous dexmedetomidine did not alter renal function in this cohort of relatively low-risk elective CABG patients but was associated with an increase in urinary output.This study was carried out in 1994-1997 and was thus not registered. Acute surgery . The pat surgery . It is a surgery , dexmedend water ,9. Moreo surgery . In an e surgery .To date, no single pharmacological regimen has conclusively proved its efficacy in preventing AKI  and any This was a double-blind, randomized, parallel-group study designed to compare dexmedetomidine, administered as a continuous intravenous (i.v.) infusion at rates needed to achieve a pseudo steady-state plasma concentration of 0.60 ng/ml, with placebo in terms of renal effects. Altogether 93 patients were screened and 87 were randomized to receive the study treatment Figure  in two s2-agonists to treat hypertension; untreated hypertension; cerebrovascular disease; type 1 diabetes mellitus or diabetes insipidus; morbid obesity; evidence or suspicion of abnormal liver function due to drug abuse or alcoholism; a history of severe allergy to drugs; and previous exposure to dexmedetomidine. Pregnant or breastfeeding women were ineligible, as were women of childbearing age not using reliable contraceptive methods. Randomization was performed by an independent statistician using random permuted blocks of 10 patients within each center. Both centers were provided with indistinguishable ampoules of dexmedetomidine and placebo to be allotted in numerical order to consecutive patients enrolled by study physicians. All patients, personnel and investigators including the persons responsible for data management and statistics were blinded to the treatment assignment. The code was opened only after recruitment and data collection of all study patients had been completed.The study was performed according to Helsinki Declaration and standards of Good Clinical Practise. The protocol and amendments were reviewed and approved by separate Ethics Committees of Turku and Helsinki Universities and the Finnish Medicines Agency. All patients provided written informed consent. Adult (aged > 21 years) patients undergoing scheduled elective CABG surgery at the two study centers were candidates for inclusion in the study if they had normal renal function and serum creatinine (S-Crea) < 115 μmol/l, were of American Society of Anesthesiologists (ASA) physical status III or IV, and met the general participation criteria for the study. Patients were not admitted to the study if they had left main coronary artery occlusion > 50%, valvular dysfunction necessitating operation, hemodynamically significant arrhythmia, or left ventricular ejection fraction < 40%. Other exclusion conditions included: treatment with angiotensin-converting enzyme inhibitors or diuretics; use of alphaAll patients received zopiclone 7.5 mg orally the night before surgery. Oral lorazepam 4-6 mg was administered 2 h before induction of anesthesia.-1 · h-1 until commencement of ECC. Baseline hemodynamic and blood chemistry measurements were obtained after at least 5 min of stabilisation after catheterisation. Anesthesia was induced with 30 μg/kg fentanyl given as a 5-min i.v. infusion and co-administered with i.v. lorazepam 2 mg. Pancuronium 0.1 mg/kg was then administered i.v. and a urinary catheter introduced. All urine passed during the 48 h after catheter insertion was collected.Patients were instrumented upon arrival in the operating theater, including the introduction of a pulmonary artery catheter. Ringer's acetate was infused intravenously at 10 ml · kgStudy drug infusion was commenced immediately after anesthesia induction, and continued until 4 h after arrival in the intensive care unit (ICU). To reach and maintain a constant pseudo steady-state plasma concentration of 0.60 ng/ml of dexmedetomidine a five-step infusion of 4.0 μg/ml dexmedetomidine or placebo with the following decreasing infusion rates was used: 39.0 ml/h for 20 min, 24.5 ml/h for 40 min, 14.0 ml/h for 60 min, 10.5 ml/h 120 min and 7.0 ml/h from that on. The dosing was based on our previous experience with dexmedetomidine in CABG patients .-1 · m-2 and reduced to 1.8 l min-1 · m-2 during hypothermia. Cold crystalloid potassium cardioplegic solution was used as required to maintain asystole.CABG was performed under ECC using moderate systemic hypothermia , a standardized protocol and mannitol 150 mg/ml 100 ml added to ECC priming. ECC was initiated at a flow rate of 2.4 l min-1 · min-1 until rewarming . Isoflurane was administered into the perfusion unit at an inspiratory concentration of 0.2%. Patients were ventilated with 40-60% oxygen in air with a target arterial CO2 tension of 35-45 mmHg (not corrected to the patient's temperature) and oxygen saturation (SaO2) > 95%. Fentanyl and isoflurane were discontinued after skin closure.Intraprocedural anaesthesia was maintained with fentanyl 0.1 μg · kgCardiovascular parameters and clinical signs were used as indicators of anesthetic depth. Specific provisions and interventions were developed in the study protocol to maintain a sufficient level of anesthesia and acceptable hemodynamics, to prevent tachycardia, bradycardia, hypertension or hypotension lasting 1 min or more and manage low urinary output intraoperatively and postoperatively. These criteria and the respective therapeutic interventions are summarized in table The primary endpoint of the study was renal function assessed by determination of creatinine clearance at 12-24 h prior to surgery, and 0-24 h and 24-48 after urinary catheter insertion after induction of anesthesia. Creatinine clearance was defined as (U-Crea) × (urine output)/S-Crea, where U-Crea and S-Crea represent urinary and serum creatinine, respectively.Creatinine clearance from 30 min urine output at 4-h intervals was quantified. Cumulative and 4-hourly urinary output was examined. Fractional sodium excretion (dU-Na) and fractional potassium excretion (dU-K) were determined at the same time as creatinine clearance. Urine samples for determination of urinary potassium (U-K), urinary sodium (U-Na), U-Crea, urinary glucose (U-Gluc) and urinary osmolality (U-Osmol) were taken after urinary catheter insertion, 1 min after aortic cross clamp removal and 24 and 48 h after urinary catheter insertion. In addition samples for determination of U-Na and U-Crea were taken at 4 h intervals until 24 h from catheter insertion.Plasma epinephrine and norepinephrine concentrations were determined using high-performance liquid chromatography with coulometric detection .Forty patients per treatment group were needed to get a 80% power to detect a 35% difference between the treatment groups with a 5% (two-sided) type I error rate and assuming a standardized effect size (expected effect size divided by SD of the outcome variable) of 0.63. Assuming a 10% drop-out rate, the final sample size was set at 88 patients (44/group).The analysis of renal function was based on patients meeting the following five criteria of evaluation: (i) study drug infusion commenced within 30 min after anesthesia induction and continued intraoperatively and 2-4 h postoperatively; (ii) preoperative and one other perioperative 24 h creatinine clearance value obtained; (iii) no reoperation during the study period; (iv) no development of myocardial dysfunction requiring inotropes or intra-aortic balloon pump support; and (v) extubation within 24 h post-CABG. A supplementary analysis of creatinine clearance was also carried out, based on the intention-to-treat (ITT) population of 87 patients.Creatinine clearance was analyzed using repeated-measures ANOVA with treatment and center as grouping factors and time as a within factor. Perioperative dU-Na, total and cumulative urine volume, S-Crea, U-Crea, serum and urinary electrolytes  and blood glucose were examined in a similar way. The Cochran-Mantel-Haenszel (CMH) test was used for urinary glucose, requirement, plasma catecholamines and requirements for postoperative cardiac pacing.The study was carried out in years 1994-1997 and prematurely terminated due to slow recruitment rate towards the end of the study. After opening the study code it turned out that a total of 93 Caucasian patients had been screened for eligibility, of which 87 had been randomized: 44 to receive dexmedetomidine and 43 to receive placebo. Twenty-one of the randomized patients  did not fulfill the evaluation criteria. Analysis of creatinine clearance was thus based on 66 evaluable patients . The disposition of patients is illustrated in figure Table -1 · 1.73 m-2 in the first 24 h after catheter insertion and from -11.34 to 13.44 ml · min-1 · 1.73 m-2 in the second 24 h (i.e. 24-48 h after catheter insertion). Analysis based on ITT population revealed that perioperative creatinine clearance (0-24-48 h) increased in both groups similarly to evaluable patients with a significant time effect (p < 0.001). There was no significant difference between the groups (p = 0.77). Mean maximal increases were 12.4% and 20.4% in the dexmedetomidine and placebo groups, respectively, with no significant difference between groups. The 95% CIs for differences between the treatment groups were from -15.28 to 8.88 ml · min-1 · 1.73 m-2 in the first 24 h after catheter insertion and from -9.05 to 15.38 ml · min-1 · 1.73 m-2 24-48 h after catheter insertion.Perioperative creatinine clearance increased in both groups during the study period 0-24-48 h), with a significant time effect (p < 0.001) but without significant difference between the groups (p = 0.93)  without the effect of time or treatment. Likewise, no treatment effect or effect of time in the ANOVA was noticed, but there was a treatment-time interaction for U-K (p = 0.0027). No effect of time, treatment or treatment-time interaction for U-Na was found. There was also a treatment effect on U-K (p = 0.0027 dexmedetomidine vs. placebo) at 48 h after catheter removal but not at earlier times.Perioperative S-Crea was relatively stable, with no significant differences between the groups Figure . U-Crea CMH test showed a significant difference between the groups in perioperative epinephrine concentrations (p < 0.001) although the concentrations were quite similar in both treatment groups Figure . Plasma Although urinary output increased and plasma norepinephrine decreased in these relatively low-risk elective CABG patients during dexmedetomidine treatment, we were not able to demonstrate a significant dexmedetomidine-related benefit in terms of renal function as evaluated with creatinine clearance. In spite of the fact that the targeted number of evaluable patients was not reached, the confidence intervals for group differences in creatinine clearance clearly demonstrate that our sample size was adequate for a negative (no difference) conclusion. Because of smaller than anticipated variance in the primary endpoint, we can exclude a larger than 15% difference between the groups. We argue that such a small difference would hardly have clinical significance. It should also be noted that the protocol stipulated the attainment and maintenance of a dexmedetomidine plasma level of 0.6 ng/ml, which is at the lower end of the preferred therapeutic concentration . NeverthPostoperative AKI is a relatively rare complication of CABG but has a profoundly disadvantageous impact on short-term (< 30 day) prognosis. In-hospital mortality rates of 40% and more have been reported in several series of patients, compared with rates of around 1% in patients without kidney insufficiency -17. LoefIn the present study, creatinine clearance was determined by direct measurement. We are supportive of assertion of Holzmann and colleaques that indices of clearance may be preferable to serum creatinine levels as an indicator of renal function . Others 2-adrenergic agonists in clinical settings have not been studied thoroughly so far. Pretreatment with the archetypal alpha2-adrenergic agonists, clonidine, has shown preventive effects of renal function expressed as creatinine clearance after cardiac surgery [2-adrenergic agonist than dexmedetomidine and has somewhat different pharmacodynamic profile [Renal effects of alpha surgery . Neverth profile .2-adrenergic agonists. Our results regarding norepinephrine support this assumption as plasma norepinephrine was reduced at various time points during the study. However, these indications of reduced sympathetic activity might have also an influence on the greater blood loss in the dexmedetomidine group compared to the placebo group, since high adrenergic output favours thrombosis. On the other hand, the relatively low number of patients and the nature of the surgery might also predispose evaluation of blood loss to type I error. More postoperative pacing was needed in dexmedetomidine group which most probably is attributable to the direct effect of dexmedetomidine on cardiac conduction times [Attenuated response of sympathetic nervous system to CABG has been suggested as a potential benefit of alphaon times .2-adrenergic action [The enhancement of urinary output with dexmedetomidine during the first 24 h after catheter insertion is favourable for dexmedetomidine, though it does not necessarily indicate a preserved renal function. Nevertheless, there was an increase of creatinine clearance from baseline in both groups and the increase was 11% higher in the placebo group, though this difference did not reach statistical significance. The increase seen in both treatment groups in creatinine clearance might have been an artifact resulting from diurnal variation  and morec action . We did The fact that we included patients at a relatively low risk of AKI may limit the interpretation of the results. In addition, we had a high drop-out rate of 21 patients due to the complexity of the study protocol. Nevertheless, all patients were included in the ITT analyses for the main end-point which were essentially the same as the per-protocol analyses.This study showed that the use of intravenous dexmedetomidine did not alter renal function in this cohort of relatively low-risk elective CABG patients although it was associated with an increase in urinary output as compared to placebo. Further trials are warranted to elucidate the effect of dexmedetomidine in high-risk patients.Defined in the text where first used.This study was funded by Orion-Pharma (Finland). The institutions involved received an unlimited research grant from Orion-Pharma, but none of the investigators received individual fees for this study. Dr. Aantaa reports that he has been a paid consultant for both Orion Pharma and Abbott Laboratories, the co-developers of dexmedetomidine, and has received research grants from Orion Pharma. Dr. Aantaa is one of the three original authors of the dexmedetomidine patent "Use of dexmedetomidine for intensive care unit sedation". The worldwide patent rights have been transferred to Orion Pharma and Abbott Laboratories. Dr. Jalonen reports that he has received research grants from Orion Pharma for studies on dexmedetomidine and other drugs. Dr. Salmenperä reports that he has received research grants from Orion Pharma for studies on levosimendan. Dr. Scheinin reports that he has been a former employee and a paid consultant for Orion Pharma. Doctors Leino, Hynynen, Kuitunen, Valtonen, Perttilä, Heikkilä and Savunen declare that they have no competing interests.1) Department of Anaesthesiology, Intensive Care, Emergency Care and Pain Medicine, Turku University Hospital, Turku, Finland; 2) Department of Anesthesia and Intensive Care Medicine, Helsinki University Hospital, Jorvi Hospital, Espoo, Finland; 3) Department of Anesthesiology and Intensive Care, Helsinki University Hospital, Helsinki, Finland; 4) Department of Pharmacology, University of Turku, Turku, Finland; 5) Department of Anesthesiology, Hospital Pulssi, Turku, Finland; 6) Department of Surgery, Turku University Hospital, Turku, FinlandKL participated in interpretation of results and writing. MH participated in the design of the study, performance of the study, interpretation of results and writing. JJ participated in the design of the study, performance of the study, interpretation of results and writing. MS participated in the design of the study, performance of the study, interpretation of results and writing. HS participated in interpretation of results and writing. RA participated in the design of the study, interpretation of results and writing. The members of the Dexmedetomidine in Cardiac Surgery Study Group  participated in performance of the study, interpretation of results and writing. All authors read and approved the manuscript and gave their intellectual contribution for it.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/11/9/prepub


Intermittent fasting  facilitates weight loss and improves coronary heart disease (CHD) risk indicators. The degree to which weight loss can be enhanced if IF is combined with calorie restriction (CR) and liquid meals, remains unknown.This study examined the effects of IF plus CR (with or without a liquid diet) on body weight, body composition, and CHD risk.Obese women (n = 54) were randomized to either the IFCR-liquid (IFCR-L) or IFCR-food based (IFCR-F) diet. The trial had two phases: 1) 2-week weight maintenance period, and 2) 8-week weight loss period.P = 0.04) in the IFCR-L  versus the IFCR-F group . LDL peak particle size increased (P < 0.01), while heart rate, glucose, insulin, and homocysteine decreased (P < 0.05), in the IFCR-L group only.Body weight decreased more (P = 0.04) in the IFCR-L group (3.9 ± 1.4 kg) versus the IFCR-F group (2.5 ± 0.6 kg). Fat mass decreased similarly (P < 0.0001) in the IFCR-L and IFCR-F groups . Visceral fat was reduced (P < 0.001) by IFCR-L (0.7 ± 0.5 kg) and IFCR-F (0.3 ± 0.5 kg) diets. Reductions in total and LDL cholesterol levels were greater (These findings suggest that IF combined with CR and liquid meals is an effective strategy to help obese women lose weight and lower CHD risk. Coronary heart disease (CHD) remains the number one of killer of women in the United States. Weight It is well known that many obese subjects are unable to adequately estimate portion sizes. This inAccordingly, the objective of the present study was to examine the effects of an IF protocol combined with CR on body weight, body composition, and CHD risk factors in obese women. Whether the addition of liquid meal replacements to this protocol would result in greater weight loss and more pronounced CHD risk reduction was also assessed.2, waist circumference >88 cm, weight stable for 3 months prior to the beginning of the study (i.e. <5 kg weight loss or gain), non-diabetic, no history of cardiovascular disease, no history of cancer, sedentary or lightly active for 3 months prior to the beginning of the study ), non-smoker, not claustrophobic, and not taking weight loss, lipid-lowering, or glucose-lowering medications. Peri-menopausal women were excluded from the study, and post-menopausal women (defined as absence of menses for 2 y) were required to maintain their current hormone replacement therapy regimen for the duration of the study. The experimental protocol was approved by the Office for the Protection of Research Subjects at the University of Illinois, Chicago, and all volunteers gave written informed consent to participate in the trial.Subjects were recruited by means of advertisements placed on and around the University of Illinois campus in downtown Chicago. A total of 77 individuals responded to the advertisements, but only 60 were deemed eligible to participate after the preliminary questionnaire, body mass index (BMI) and waist circumference assessment Figure. Key incRandomization was performed by way of a stratified random sample. The sample frame was divided into strata based on BMI and age. Subjects from each stratum were then randomly assigned to either the IFCR-liquid diet (IFCR-L) group (n = 30) or IFCR-food based diet (IFCR-F) group (n = 30). The 10-week trial consisted of two dietary phases: 1) a 2-week baseline weight maintenance period, and 2) an 8-week weight loss period.Before commencing the 8-week weight loss intervention, each subject participated in a 2-week baseline weight maintenance period. During this period, subjects were requested to maintain a stable weight and continue eating their usual diet.IFCRL subjects (n = 30) consumed a calorie-restricted liquid diet for the first 6 days of the week, and then underwent a fast on the last day of the week . The liquid diet (during the CR period) consisted of a liquid meal replacement for breakfast  and a liquid meal replacement for lunch . All liquid meal replacements were provided to the subjects in powder-form in premeasured packets . At dinnertime, IFCR-L subjects consumed a 400–600 kcal meal. Food was not provided to the subjects for the dinnertime meal. Instead, subjects met with a Registered Dietician weekly to learn how to make healthy eating choices that are in compliance with the National Cholesterol Education Program Therapeutic Lifestyle Changes (TLC) diet . In following this regimen, each subject was calorie restricted by 30% of their baseline needs. IFCRF subjects (n = 30) consumed a calorie-restricted food-based diet for the first 6 days of the week, and then underwent a fast on the last day of the week . IFCR-F subjects ate 3 meals per day in accordance with the TLC diet guidelines. Food was not provided to the subjects. Instead, subjects met with a Registered Dietician weekly to learn how to make healthy eating choices that were in compliance with the TLC diet. Subjects were instructed to eat approximately 240 kcal for breakfast, 240 kcal for lunch, and 400–600 kcal for dinner. In following this regimen, each IFCR-F subject was calorie restricted by 30% of their baseline needs.Following the baseline period, subjects participated in the IFCR-L or IFCR-F protocol for 8 weeks. Energy requirements were measured using the Mifflin equation. IFCRL sA 7-d food record was used to assess adherence to the diets. Subjects completed the food records at week 3 and 10. The Registered Dietician provided 15 min of instruction to each participant on how to complete the food records. These instructions included verbal information and detailed reference guides on how to estimate portion sizes and record food items in sufficient detail to obtain an accurate estimate of dietary intake. All dietary information from the food records was entered into the food analysis program, Nutritionist Pro  by a single trained operator to alleviate inter-investigator bias. In addition, IFCR-L subjects were provided with a checklist each day to monitor: 1) adherence to the liquid meal protocol, and 2) adherence to the fast day regimen. IFCR-F subjects were also given a checklist to monitor their adherence to the fast day regimen. If an IFCR-L or IFCR-F subject consumed more that 100 kcal on the fast day , the subject was considered non-adherent with the fast day protocol.Changes in daily energy expenditure associated with alterations in physical activity habits were quantified by the use of a pattern recognition monitor (Sense Wear Mini (SWM), Bodymedia, Pittsburgh, PA). Subjects wore the lightweight monitor on their upper arm for 7 d at week 3 and 10 of the trial. The SWM is a wireless multi-sensor activity monitor that integrates motion data from a triaxial accelerometer along with several other physiological sensors . The data was analyzed using Bodymedia Software V.7.0, algorithm V.2.2.4 .2. Fat mass and fat free mass were assessed by dual energy X-ray absorptiometry (DXA) at weeks 1, 3 and 10 . Waist circumference was measured by a flexible tape to the nearest 0.1 cm, midway between the lower costal margin and super iliac crest during a period of expiration. Abdominal visceral adipose tissue and subcutaneous adipose tissue volumes were quantified using magnetic resonance imaging (MRI) at week 3 and 10[2) was performed using HippoFat image analysis software[3, the density of adipose tissue, to obtain total visceral adipose tissue mass (kg) and total subcutaneous adipose tissue mass (kg)[Body weight measurements were taken to the nearest 0.5 kg at the beginning of each week in light clothing and without shoes using a balance beam scale . Height was assessed using a wall-mounted stadiometer to the nearest 0.1 cm. BMI was assessed as kg/msoftware. VisceraTwelve-hour fasting blood samples were collected between 6.00 am and 9.00 am at weeks 1, 3 and 10. The subjects were instructed to avoid exercise, alcohol, and coffee for 24 h before each visit. Blood was centrifuged for 10 min at 520 × g at 4°C to separate plasma from red blood cells and was stored at −80°C until analyzed.Plasma total cholesterol, direct LDL cholesterol, HDL cholesterol, and triglyceride concentrations were measured in duplicate by enzymatic kits . The interassay coefficient of variations (CV) for total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were 2.4%, 3.7%, 4.0%, and 3.5%, respectively. LDL particle size was measured by linear polyacrylamide gel electrophoresis . High-reBlood pressure and heart rate were measured in triplicate each week using a digital automatic blood pressure/heart rate monitor  with the subject in a seated position after a 10-min rest. Fasting plasma glucose was measured in duplicate at week 1, 3, and 10 with a glucose hexokinase reagent kit . Insulin, C-reactive protein (CRP), homocysteine, adiponectin, and leptin were assessed in duplicate at week 1, 3, and 10 by ELISA .Results are presented as mean ± SEM. An independent samples t-test was used to test baseline differences between groups. Repeated-measures ANOVA was performed (taking time as the within-subject factor and diet as the between-subject factor) to assess differences between groups over the course of the study. Post-hoc analyses were performed using the Tukey test. Differences were considered significant at P < 0.05. All data was analyzed using SPSS software .Of the 30 subjects that were randomized to the IFCR-L group, 1 subject dropped out due to scheduling conflicts and 1 subject dropped out because they could not adhere to the diet , adherence to the liquid meal protocol was 92 ± 3% in the IFCR-L group over the course of the 8 weeks. IFCR-F subjects achieved their energy restriction goal on 80 ± 2% of the CR days during each week. As for fast day compliance, there were no differences between groups (P = 0.91) . The degree of CR achieved during the weight loss period by the IFCR-L group (29 ± 3%) was greater (P < 0.05) than that achieved by the IFCR-F (22 ± 4%). The macronutrient composition of the IFCR-L diet  did not differ from that of the IFCR-F diet  during the 8-week intervention. Alterations in physical activity habits were quantified by the use of a pattern recognition monitor (i.e. an accelerometer). There were no differences in activity energy expenditure between baseline and post-treatment in the IFCR-L group , and the IFCR-F group .2, respectively, in the IFCR-L and IFCR-F groups. Fat mass decreased (P < 0.0001) in the IFCR-L and IFCR-F groups by 2.8 ± 1.2 kg and 1.9 ± 0.7 kg, respectively. Fat free mass remained unchanged throughout the course of the trial in both groups. Visceral fat was reduced (P < 0.001) in the IFCR-L (0.7 ± 0.5 kg) and IFCR-F (0.3 ± 0.5 kg) groups after 8 weeks of treatment. Abdominal subcutaneous fat was not affected by either intervention. There were no differences between groups for fat mass, fat free mass, visceral adipose tissue, or abdominal subcutaneous adipose tissue at any time point. FigureChanges in body weight and body composition are reported in FigureIFCR-F 0. ± 0.5 kgPlasma lipids did not change during the baseline period in either the IFCR-L or IFCR-F group. Changes in plasma lipid concentrations during the weight loss period are displayed in FigureThere were no changes in CHD risk parameters during the weight maintenance period. Changes in CHD risk indices during the weight loss period are displayed in TableOur findings show, for the first time, that the combination of IF plus CR is an effective means of reducing body weight, fat mass, and visceral fat mass in obese women. This novel regimen also decreased key indicators of CHD risk, such as LDL cholesterol, triglycerides, and the proportion of small LDL particles. When liquid meal replacements were incorporated into the IFCR regimen, greater reductions in body weight and indicators of heart disease risk were noted.Studies of IF in human populations are very limited. To our knowledge, only two studies have examined the effect of IF on body weight,13. In aBeneficial modulations in key lipid risk factors were also observed by both diets. For instance, total and LDL cholesterol decreased in the IFCR-L group  and IFCR-F group , with greater changes noted for IFCR-L. In contrast, only the IFCR-L group experienced reductions in triglycerides (17% from baseline). The proportion of small LDL particles was also decreased by both the IFCR-L group  and IFCR-F group . However, increases in LDL peak particle size and the proportion of large LDL particles, were only noted in the IFCR-L group. Taken together, these results suggest that IF combined with CR is an effective means of improving lipid profile in a short-term (8 week) intervention. We also show that adding a liquid diet component may enhance this lipid-lowering effect. The greater decreases in plasma lipids by the IFCR-L diet is most likely due to the greater weight loss noted in this group. For every kg of body weight loss, LDL cholesterol is estimated to decrease by 2 mg/dl. Since tModulations in other CHD risk parameters were also more pronounced in the IFCR-L group compared to the IFCR-F group. For instance, fasting plasma glucose and insulin were only decreased by the liquid intervention, suggesting that this diet therapy may benefit glycemic control. Heart rate and homocysteine concentrations were reduced solely in the IFCR-L group. Leptin concentrations, on the other hand, were lowered by both diets. The decreases in leptin are most likely mediated by the reductions in fat mass and visceral fat mass observed in both groups. Leptin This study is limited in that it did not tease apart the effect of IF and CR on body weight and CHD risk. Thus, the independent contributions of the IF diet versus the CR diet on these various parameters, are not known. In order to answer these key questions, a future study should be performed that compares the effect of IF combined with CR, to that of IF alone, and CR alone. An additional limitation of the study was that it did not carefully control for food intake by providing food-based meals to the intervention groups . If meals were provided, a more precise measurement of energy restriction and dietary adherence could have been obtained. The study is also limited in that it employed HippoFat software to quantify visceral fat mass from MRI images. This software is limited in that it underestimates visceral fat and overestimates subcutaneous fat, particularly in larger individuals. AnotherIn summary, these findings suggest that IFCR may be effective for reducing body weight, visceral fat mass, and CHD risk in obese women. We also report that incorporating liquid meal replacements into an IFCR regimen may facilitate greater weight loss and lipid-lowering. From a clinical standpoint, we would recommend this diet to individuals who wish to boost the weight loss they see with IF, by adding a daily CR regimen. This combination may also help reduce the boredom typically associated with attempting only one dietary plan. Although these short-term findings are promising, the long-term effects of this novel diet strategy still require confirmation in a large-scale human trial.Krista Varady has a consulting relationship with the sponsor of the research, lsagenix, LLC.MCK conducted the clinical trial, analyzed the data, and assisted with the preparation of the manuscript. CMK assisted with the conduction of the clinical trial and performed the laboratory analyses. SB and JFT performed the laboratory analyses, and assisted with data analyses. KAV designed the experiment and wrote the manuscript. All authors read and approved the final manuscript.This study was funded by Isagenix LLC., Chandler, AZ


Subsequent palladium-catalyzed reactions  delivered, after cleavage, the desired molecules in moderate to good yields over four steps. The scope and limitations are presented.The synthesis of diverse substituted indole structures on solid supports is described. The immobilization of nitrobenzoic acid onto Merrifield resin and the subsequent treatment with alkenyl Grignard reagents delivered indole carboxylates bound to solid supports. In contrast to results in the liquid phase,  Indoles are heterocyclic structures of unquestionable importance. It is well recognized that the indole moiety is a privileged structural motif found in numerous natural products, such as alkaloids or peptides, and various synthetic compounds –2. Moreoortho-substituted nitroarenes gave good yields of the indoles. Despite the interest in new indoles + refers to the molecular ion. Elemental analysis: Elementar Vario EL. Routine monitoring of reactions was performed by using silica-gel-coated glass plates , which were analyzed under UV light at 254 nm and/or dipped into a solution of molybdato phosphate  or potassium permanganate (0.45 g of potassium permanganate and 2.35 g of sodium carbonate in 90 mL of water) and heated with a heat gun. Solvent mixtures are understood as volume/volume. Solid materials were powdered. Solvents, reagents and chemicals were purchased from Aldrich, Fluka, ABCR, Acros, Merck and Lancaster. Solvents, reagents and chemicals were used as purchased unless stated otherwise. Merrifield resin , was purchased from CalBiochem-NovaBiochem.General washing procedure for resins: After reaction the resins are subsequently washed according to the following procedure:  three times,  once,  twice.GP 1 - Immobilization of benzoic acids on Merrifield resin: In a three-necked round-bottom flask equipped with a mechanical stirrer, 5 equiv of cesium carbonate are suspended in DMF (mL/mmol) and are stirred for 30 min at 50 °C. Next, 5 equiv of benzoic acid are added and the mixture is stirred for another 30 min. Afterwards, one equiv of Merrifield resin is added and the suspension is stirred for 24 h at 50 °C. After being cooled down to room temperature the resin is filtered off and first washed with water, then treated according to the general washing procedure, and dried in high vacuum. The loading of the resin was calculated according to the nitrogen values of the elemental analysis.GP 2 - Bartoli-indole synthesis: Under an argon atmosphere, one equiv of the resin is suspended in dry THF (0.1 mmol/mL), cooled down to −40 °C and three equiv of the Grignard reagent are added, while the color of the mixture changes to orange-red. The reaction mixture is allowed to warm to 0 °C, and then a saturated aqueous solution of ammonium chloride is added. The resin is filtered off, and first washed with water, then according to the general washing procedure, and dried in high vacuum. The loading of the resulting resin was calculated as if complete conversion had taken place.GP 3 - Suzuki reaction: Under an argon atmosphere, one equiv of the respective 7-bromo-1H-indole-6-carboxymethyl-polystyrene is suspended in DMF (0.1 mmol/mL) together with 0.10 equiv of tetrakis(triphenylphosphine)palladium and two equiv of boronic acid. An aqueous solution of sodium carbonate  is added and the mixture agitated for two days at 80 °C. After cooling down to room temperature, 10 mL of a 25% aqueous solution of ammonium acetate are added, the resin is filtered off, washed according to the general washing procedure, and dried in high vacuum. The loading of the resulting resin was calculated as if complete conversion had taken place.GP 4 - Sonogashira–Hagihara reaction: Under an argon atmosphere, one equiv of the respective 7-bromo-1H-indole-6-carboxymethyl-polystyrene is suspended in DMF (0.1 mmol/mL) together with 10.0 mol % bis(triphenylphosphine)palladium(II) chloride, 15.0 mol % copper(I) iodide and one equiv of triphenylphosphine. Then, two equiv of triethylamine and 2.5 equiv of 4-ethynylanisole are added and the mixture is agitated for two days at 80 °C. After cooling down to room temperature, 10 mL of a 25% aqueous solution of ammonium acetate are added, the resin is filtered off, washed according to the general washing procedure, and dried in high vacuum. The loading of the resulting resin was calculated as if complete conversion had taken place.GP 5 - Stille reaction: Under an argon atmosphere, one equiv of the respective 7-bromo-1H-indole-6-carboxymethyl-polystyrene is suspended in DMF (0.1 mmol/mL) together with 10.0 mol % bis(triphenylphosphine)palladium(II) chloride, 15.0 equiv of lithium chloride and one equiv of triphenylphosphine. Then, three equiv of tributyl(vinyl)tin are added and the mixture is agitated for two days at 80 °C. After being cooled down to room temperature, 10 mL of a 25% aqueous solution of ammonium acetate are added, and the resin is filtered off, washed according to the general washing procedure, and dried in high vacuum. The loading of the resulting resin was calculated as if complete conversion had taken place.GP 6 - General cleavage protocol: To one equiv of the resin in dry THF (0.1 mmol/mL), a solution of 30% sodium methoxide in MeOH (2.00 mL/mmol of resin) is added and the mixture is agitated for three hours at room temperature. The resin is filtered off, and the filtrate is hydrolyzed with water and subsequently extracted with ethyl acetate three times. After being dried over magnesium sulfate, the solvent is removed under reduced pressure and the residue is purified by column chromatography by using cyclohexane/ethyl acetate, 3:1 as eluent.File 1Experimental details.